1 | INTRODUCTION
Cytosolic protein delivery promisingly expands therapeutic
possibilities[1,2]. The delivery of active
proteins replaces disease-causing deficient or dysfunctional proteins
that are important for essential cellular
events[3]. Recently, the delivery of engineered
proteins, such as CRISPR/Cas9, has been reported to artificially
modulate genomic information, thereby opening a new window for
therapeutic applications[4]. However, the
development of precise and efficient technologies for cytosolic protein
delivery remains challenging. As proteins are generally
membrane-impermeable owing to their macromolecular nature and
hydrophilic properties, most proteins cannot spontaneously enter the
cells through cell membranes. Cell-permeable carriers consisting of
peptides[5],
polymers[4,6-8],
liposomes[9,10], and
nanoparticles[11] have been actively employed to
transport proteins inside cells. Although a few carriers have achieved
direct cytosolic delivery of proteins by fusion with cellular plasma
membranes[10,11], most conventional carriers are
usually taken up via endocytosis. Without being released from endosomes,
cargo proteins are degraded in lysosomal compartments before being
functional in the cytosol[2]. Therefore,
technologies for endosomal escape have been developed by utilizing the
proton sponge effect of pH-buffering agents, such as
poly(ethyleneimine)[12] and a charge conversion
block polymer[6], by fusion of a carrier with the
endosomal membrane[13] and by destabilization of
the endosomal membrane with endosome-disruptive
peptides[14,15],
polymers[7,16],
nanoparticles[11,17], and
photosensitizers[18-21]. Among these technologies,
photochemical approaches offer photo-triggered spatiotemporal delivery
of cargo proteins into the cytosol[19-22].
Selective cytosolic protein delivery at the desired timing and sites
holds promise for safer and more effective
therapies[1,22]. However, the light used during
most photochemical reactions generally does not penetrate through the
deeper areas of the body because of its low permeability in living
tissues[23].
Ultrasound readily penetrates deep into the interior of the body in a
noninvasive manner [24]. Using focusing
techniques, ultrasound exposure can be limited to a localized region,
leading to selective exposure at the target site. Accordingly,
ultrasound-responsive carriers for protein delivery have been reported
to be promising non-invasive tools for spatiotemporal administration of
proteins in deeper areas of the body[25-27].
However, no ultrasound-responsive carriers for cytosolic protein
delivery have been reported till date. A method for ultrasound-induced
drug release from endosomes has recently been
reported[28]. In a pioneering study, liposomes
containing fluorescent dyes and perfluorocarbon nano-droplets
(phase-change nano-droplets, PCNDs) were introduced into living cells by
folate-mediated endocytosis, and subsequent ultrasound-induced
vaporization of PCNDs led to endosome rupture, thereby achieving escape
of the dyes from the endosomes. In this study, we envisioned that
endosomal rupture through vaporization of PCNDs could be applied to
ultrasound-induced cytosolic protein delivery. In our previous reports,
PCNDs conjugated with an anti-epiregulin (EREG) antibody (named as 9E5)
were taken up by high-EREG-expressing cancer cells via
endocytosis[29] and were confirmed to vaporize
inside the cells by exposure to ultrasound[30,31].
Therefore, 9E5-conjugated PCNDs were used as carriers for cytosolic
protein delivery. Cargo proteins were conjugated with PCNDs via a
bio-reductively cleavable disulfide linker. Protein-conjugated PCNDs
were introduced into living cells via 9E5-mediated endocytosis, and
after exposure to ultrasound, the cytosolic delivery of an enzyme and a
cytotoxic protein was examined (Figure 1).